Surgery Today Jpn. J. Surg. (1992) 22:351-356
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SURC~RYTODAY © Springer-Verlag 1992
Expression of the Cell Surface Antigen Detected by the Monoclonal Antibody A7 in Pancreatic Carcinoma Cell Lines EIGO OTSUJI, 1 TOSHIHARU YAMAGUCHI,1 NOZOMI YAMAGUCHI,2 KUNIHIKO KOYAMA,3 JIRO IMANISHI,2 NOBUKI YAMAOKA,2 and TOSHIO TAKAHASHIt 1The First Department of Surgery, and the Departments of 2Microbiology and 3Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji Agaru Kajiicho 465, Kamigyo-ku, Kyoto 602, Japan
Abstract: In a previous study, we used a murine monoclonal antibody, AT, against human colon carcinoma as a drugcarrier to treat colorectai cancer. ~ In the present study, we found that MAb A7 also reacted immunohistochemically with 73% of human pancreatic carcinoma cell lines, with the A7 antigen mainly being detected on the cell surface. However, the A7 antigen was found in only 9% of the spent media of these human pancreatic carcinoma call lines by ELISA. On the other hand, the positive incidence of CA19-9, POA, ferritin, CEA, DU-PAN-2 and SLX in those spent media was 100%, 64%, 64%, 55%, 55% and 36%, respectively. These results suggest that the A7 antigen may only rarely be shed into the sera of pancreatic cancer patients, in which case MAb A7 could be a suitable drug-carrier in targeting chemotherapy for pancreatic cancer patients. Key Words: monoclonal antibody A7, pancreatic tumor
marker, pancreatic carcinoma cell line, immunotargeting chemotherapy
Introduction
A number of antigens associated with pancreatic carcinoma, such as carcinoembrionic antigen (CEA), pancreatic oncofetal antigen (POA), ferritin, DUPAN-2, CA19-9 and sialyl Le x antigen (SLX), have been identified using polyclonal or monoclonal antibodies. 2-8 Although most antibodies induced by these antigens were not primarily produced by immunization with pancreatic carcinomas, they have been detected in pancreatic carcinoma by analyses using various kinds of
Reprint requests to: E. Otsuji (Received for publication on Jan. 8, 1991; accepted on May 1, 1992)
monoclonal antibodies. Antibodies to these antigens have been used clinically for the serological diagnosis of pancreatic cancer since large amounts of these antigens are shed or released from cancer cells into serum. For targeting chemotherapy, however, a large number of antigens in serum is disadvantageous because the administered antibody, conjugated to an anticancer agent, binds to the antigen in the serum before it arrives at the tumor. In our laboratory, the monoclonal antibody A7 (MAb) was produced against a human colon carcinoma and conjugated covalently with the anticancer antibiotic, neocarzinostatin (NCS; Kayaku, Japan). The monoclonal antibody-drug conjugate, A7-NCS, 9 was then used clinically to treat patients with colorectal carcinoma. 1 There is little information available on the treatment of pancreatic cancer with a monoclonal antibody. A7NCS might be useful for the targeting chemotherapy of pancreatic cancer if MAb A7 reacts with pancreatic carcinoma at a high rate and if large amounts of the antigen are not shed from pancreatic carcinoma cells. We describe herein the reactivity of MAb A7 with 11 human pancreatic carcinoma cell lines and the cytochemical and immunological characterization of the monoclonal antibody A7.
Materials and Methods
Cell Lines
The following cell lines were used: 11 human pancreatic carcinoma cell lines, being HPC-YO, HPC-Y1, HPCY5, HPC-Y9, HPC-Yll, HPC-Y15, HPC-Y19, HPCY25, HPC-YP, HPC-YS and HPC-YT, a human colonic carcinoma cell line, being WiDr, and a human laryngeal carcinoma cell line, being KB-2. All the
352 pancreatic carcinoma cell lines were established from ductal cell adenocarcinoma except for HPC-YO, which was established from acinal cell carcinoma. All the human pancreatic carcinoma cell lines were established by Dr. Yamaguchi of the Kyoto Prefectural University of Medicine, Japan, who also maintained and kindly donated the other cell lines. These cell lines were cultured in protein-free Ham's F-12 medium (Flow Laboratories, USA) without any other supplements, as described previously. 1°'~1
Spent Media of the Human Pancreatic Carcinoma Cells The serum-free medium was changed every 4 days and the spent medium collected aseptically. After the cellular debris was removed by centrifugation, the spent medium was stored at 4°C and concentrated by ultrafiltration using a PTGC membrane with a molecular weight cut off of 10,000 (Nippon Millipore, Japan) at 0°C. 1°'11 Finally, the concentrated spent medium was dialyzed against distilled water and lyophilized.
Monoclonal Antibody A 7 MAb A7 was produced by a hybridoma obtained after fusing spleen cells from a mouse immunized with human colon adenocarcinoma, Colon-6, with murine myeloma P3.X63.Ag8.653 cells, as described previously, lz MAb A7 was purified by the Protein A method.
Immunoperoxidase Staining of the Human Pancreatic Carcinoma Cell Lines Cultured human pancreatic carcinoma cells detached with phosphate-buffered saline (PBS) containing 2 mM EDTA were washed in PBS three times and smear specimens prepared. After fixation with cold acetone for five min, immunoperoxidase staining was performed by the avidin-biotin-peroxidase complex (ABC) method (Vector Labs, USA). 13 The carcinoma cells were then stained with MAb A7 in PBS (10pg/ ml) with the monoclonal antibodies of DU-PAN-2 (Kyowa medex, Japan), diluted to 1/300 with PBS, CA19-9 (CIS Kit) and SLX (Otsuka Assay Lab., Japan) in a spent medium of FH-6, and with the polyclonal antibodies of CEA (DAKO, Denmark, 1/500), POA (provided by Dr. Takami of the University of Teikyo, 1/500) and ferritin (Miles, Denmark, 1/500). Finally, the specimens were developed with diaminobenzidine for color, then counterstained with hematoxylin.
E. Otsuji et al.: Expression of the A7 Antigen
Cellular Localization of the Antigen Recognized by MAb A7 After the HPC-YS cells were harvested with EDTA and washed in PBS three times, they were fixed with 10% buffered formalin in PBS for one hour at room temperature. Following dehydration, they were embedded in paraffin and 6-~tm serial sections were cut. After these sections were deparaffinized and processed in alcohol by standard techniques, immunoperoxidase staining was performed by the ABC method, after which the nucleus was counterstained with hematoxylin. Normal mouse IgG was used for the control staining.
ELISA Against a Spent Media of Human Pancreatic Carcinoma Cell Lines The ELISA was performed against the spent media of 11 human pancreatic carcinoma cell lines. Thirtyfive ~tg of freeze-dried serum-free spent medium was suspended in carbonate-bicarbonate buffer (pH 9.6) and seeded in 96-well microtiter plates (Flow Laboratories, USA). After overnight incubation at room temperature, the wells were treated with 25% Block Ace (Dainippon Kagaku) in PBS. Following removal of the Block Ace, 50 pl of MAb A7 in PBS (10 ~tg/ml) was added and the wells were incubated for 1 hour at room temperature. After three washes with 0.05 per cent Tween 20-PBS, the wells were converted with peroxidase-labeled goat anti-mouse IgG + IgM (Kirkegraard and Perry Laboratories, USA). After five rinses with 0.05% Tween 20-PBS, 50pl of a substrate solution containing diethylbenzothiazolin (ABTS, Kirkegraard and Perry Laboratories, USA) was added to each well. The absorbance was read at 414nm by an automatic immunoreader (Bio Rad, USA). As a control, MAb A7 was replaced by 10 ~tg of normal mouse IgG (Funakoshi, Japan). Using the same method, ELISA was performed with the monoclonal antibodies of DU-PAN-2, CA19-9 and SLX, and the polyclonal antibodies of CEA, POA and ferritin at the same concentrations used in immunoperoxidase staining, with appropriate antisera as a control.
Biochemical Characterization of the Antigen Detected by MAb A7 on the Cultured Cells and in Their Spent Media HPC-YS cultured cells (5 × 106) were solubilized with 500 ~tl of the extraction buffer (10 mM Tris-HC1 pH.7.4, 150mM NaC1, 0.5% Nonidet-P40). After the mixture was incubated for 30 min at 0°C and centrifuged at 10,000 × g for 20 rain, the supernatant was added to the same amount of the sample buffer con-
E. Otsuji et al.: Expression of the A7 Antigen
353
Table 1. Reactivity of the antibodies to human pancreatic carcinoma cell lines
Anti-CEA Anti-POA Anti-ferritin Anti-DU-PAN-2 Anti-CA19-9 Anti-SLX A7
HPC -YO
HPC -Y1
HPC -Y5
HPC -Y9
HPC -Yll
HPC -Y15
HPC -Y19
HPC -Y25
HPC -YP
HPC -YS
HPC -YT
Total (%)
+ + + + + -
+ + + + -
+ + + + + + +
+ + + + + + +
+ + + + + +
+ + + + + +
+ + + + + + -
+ + + + +
+ + + + +
+ + + + + + +
+ + + +
11/11 (100) 9/11 (82) 10/11 (91) 6/11 (55) 11/11 (100) 6/11 (55) 8/11 (73)
taining 2% sodium dodecyl sulfate (SDS) without mercaptoethanol. The spent medium of the cultured H P C - Y 9 cells (200 gg) was mixed with 10 gl of sample buffer containing SDS without mercaptoethanol. After being boiled for 3 min, the samples were analyzed by electrophoresis in 4 - 2 0 % gradient SDS-polyacrylamide gels (Daiichi Pure Chemicals, Japan) according to the method of Laemmli. 14 Protein bands were then electrophoretically transferred to a nitrocellulose m e m b r a n e (BA85 Schleicher and Schuell, West G e r m a n y ) as described previously by Towbin 15 (LKB Electrophoretic Transfer Kit). Electroblotting was p e r f o r m e d at 60 m A for 2 hours. After the transfer, the nitrocellulose m e m brane was rinsed in Tween 20-PBS and coated with Block Ace overnight at 4°C. The m e m b r a n e was then incubated with 10 gg/ml of M A b A7 in PBS for 1 h at r o o m t e m p e r a t u r e and stained by the A B C method. As a control, the m e m b r a n e was incubated with normal mouse IgG. The lysates of the h u m a n colon carcinoma cell line WiDr, or the h u m a n larynx squamous carcinoma cell line KB-2 were electrophoresed at the same time as positive or negative controls.
Results
Immunocytochemical Reactivity of MAb A7 with the Human Pancreatic Carcinoma Cell Lines Table 1 shows the reactivity of M A b A7 with the h u m a n pancreatic carcinoma cell lines by the immunoperoxidase method in comparison with that of antibodies against other well-known t u m o r markers. M A b A7 detected the antigen in 8 of the 11 h u m a n pancreatic carcinoma cell lines (73%), namely, H P C , Y 5 , HPC-Y9, H P C - Y l l , HPC-Y15, HPC-Y25, H P C - Y P , H P C - Y S and H P C - Y T . Almost 100% of the cells in these cell lines were positively stained, except for the H P C - Y P cells, of which about 50% were stained with M A b A7. Three of the 11 cell lines (27%), being H P C - Y O , HPC-Y1 and HPC-Y19, were not stained at all by M A b A7. The anti-CEA and anti-
CA19-9 antibodies both reacted with all 11 cell lines, the anti-ferritin antibody with 10 (91%), and the antiP O A antibody with 9 (82%). Six cell lines (55%) were stained by both the anti-DU-PAN-2 and anti-SLX antibodies. All the antibodies reacted with HPC-Y5, H P C - Y 9 and HPC-YS, while HPC-Y19 stained with all the antibodies except for M A b A7.
Cellular Localization of the Antigen Detected by MAb A7 As shown in Fig. 1, the antigen was mainly detected by M A b A7 on the cell surface of HPC-YS cells with immunoperoxidase staining. The cytoplasm was only slightly stained, and the mouse I g G control did not detect the antigen.
Reactivity Against the Spent Media of the Human Pancreatic Carcinoma Cell Lines by ELISA The presence of these tumor-associated antigens in tissue culture media as determined by E L I S A is shown in Table 2. M A b A7 detected the antigen in the medium of only 1 of the 11 cell lines (9%). On the other hand, CA19-9 antigen wa~ detected in the spent media of all the cell lines (100%). P O A and ferritin were each detected in 7 (64%), and C E A and D U - P A N - 2 each in 6 spent media (55%). The positive incidence of SLX was lowest among all the well-known t u m o r markers (36%).
Analysis of the A 7 Antigen by SDS-Polyacrylamide Gel Electrophoresis and Western Blotting To determine the molecular weight (MW) of the antigen recognized by M A b A7, SDS-polyacrylamide gel electrophoresis and immunoblotting were e~rformed. In the lane where the lysates of HPC-YS and W i D r were applied, M A b A7 bound to a c o m p o n e n t with M W 42,000. In the lane with H P C - Y 9 spent medium, the binding of M A b A7 was directed against the band at M W 42,000. In the lane with the squamous
354
E. Otsuji et al.: Expression of the A7 Antigen
Fig. 1. Cellular localization of the M A b A7-defined antigen was observed by immunoperoxidase staining of formalin-fixed paraffinembedded sections of the human pancreatic carcinoma cell line, HPCYS. The nucleus was counterstained with hematoxylin and the antigen mainly detected on the cell membrane by M A b A7 (10~tg/ml) (left). Positive staining was not seen with normal mouse IgG (101ag/ml)
(right)
Table 2. Reactivity of the antibodies to the spent media of human pancreatic carcinoma cell lines
Anti-CEA Anti-POA Anti-ferritin Anti-DU-PAN-2 Anti-CA19-9 Anti-SLX A7
HPC -Y0
HPC -Y1
HPC -Y5
HPC -Y9
HPC -Yll
HPC -Y15
HPC -Y19
HPC -Y25
HPC -YP
HPC -YS
HPC -YT
+ -
+ + + -
+ + + + -
+ + + + + + +
+ + + + + .
+ + + + -
+ + + + + .
+ + + + -
+ -
+ + + + + +
+ + -
cell c a r c i n o m a cell line, K B - 2 , M A b A 7 d i d n o t b i n d to a n y c o m p o n e n t (Fig. 2). N o r m a l m o u s e I g G d i d n o t stain. W h e n 2 - m e r c a p t o e t h a n o l was a d d e d to t h e s a m p l e b u f f e r no b a n d was s e e n in a n y l a n e ( d a t a n o t shown).
Discussion This p a p e r d e s c r i b e s t h e r e a c t i v i t y o f t h e m o n o c l o n a l a n t i b o d y A 7 with 11 h u m a n p a n c r e a t i c c a r c i n o m a cell lines a n d t h e i r s p e n t m e d i a . W e u s e d a c e t o n e - f i x e d s p e c i m e n s b e c a u s e f o r m a l i n fixation a n d paraffin e m b e d d i n g s o m e t i m e s e x e r t a s t r o n g influence o n t h e p r e s e r v a t i o n o f antigenicity.16'17 M A b A 7 r e a c t e d with 7 3 % o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines. G e n e r a l l y , t h e r e a c t i v i t y o f an a n t i b o d y to a c a r c i n o m a cell line is l o w e r t h a n t h a t to c a r c i n o m a tissue which consists o f h e t e r o g e n e o u s t u m o r cells. 18 H o w e v e r , t h e p o s i t i v e i n c i d e n c e of t h e 6 w e l l - k n o w n t u m o r m a r k e r
.
.
.
.
.
Total (%) 6/11 7/11 7/11 6/11 11/11 4/11 1/11
(55) (64) (64) (55) (100) (36) (9)
a n t i g e n s in o u r h u m a n p a n c r e a t i c c a r c i n o m a cell lines was s i m i l a r to t h a t f o u n d b y o t h e r i n v e s t i g a t o r s using p a n c r e a t i c c a r c i n o m a tissues. 4'5's T h e p o s i t i v e inc i d e n c e o f M A b A 7 was l o w e r t h a n t h a t o f t h e antib o d i e s a g a i n s t CEA, C A 1 9 - 9 , ferritin a n d P O A , b u t h i g h e r t h a n t h a t o f t h e a n t i b o d i e s against D U - P A N - 2 and SLX. T h e stain!ng h e t e r o g e n e i t y o f H P C - Y P b y M A b A 7 was s i m i l a r to t h a t d e s c r i b e d in o t h e r studies. 5'19 This h e t e r o g e n e i t y m a y b e r e l a t e d to t h e stage of different i a t i o n o f t h e cells o r t h e i r p h a s e in t h e cell cycle. E L I S A was p e r f o r m e d to d e t e c t a n t i g e n s in t h e m e d i a o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines. W e u s e d a s e r u m - f r e e m e d i u m b e c a u s e it was m o r e s u i t a b l e for t h e a n t i g e n assay. 2° T h e p o s i t i v e i n c i d e n c e o f t h e w e l l - k n o w n t u m o r m a r k e r s in t h e s p e n t m e d i a o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines was also s i m i l a r to t h a t in t h e s e r a o f p a n c r e a t i c c a r c i n o m a p a t i e n t s in p r e v i o u s r e p o r t s . 4'21 T h e a n t i g e n r e c o g n i z e d b y M A b A 7 a p p e a r s to b e r a r e , o r p r e s e n t in o n l y v e r y
E. Otsuji et al.: Expression of the A7 Antigen
355
carcinoma cell lines (data not shown). Although this method is suitable for detecting the cytoplasmic antigen, the cytoplasm was stained very weakly by MAb A7. Generally, it is necessary for the antigen to be localized on the cell surface in order to detect the tumor cell in vivo. These results suggest that MAb A7 may be useful for targeting chemotherapy. Using SDS-PAGE and immunoblotting, we showed that the epitope defined by MAb A7 in the lysate of the HPC-YS cell membrane and in the spent medium of HPC-Y9 cells was located on a 42K dalton molecule. Identical results were obtained using the human colon carcinoma cell line, WiDr. This is in accordance with a previous report characterizing the antigen defined by MAb A7 on the human colon carcinoma cell line, SW 1116. 22 In the presence of mercaptoethanol in the sample buffer, however, the antigen was not detected. MAb A7 has been conjugated to NCS, and MAb A7NCS conjugate has been used to treat patients with colorectal cancer. Patients receiving the conjugate did not experience serious adverse effects, and some showed such therapeutic benefits as pain relief and tumor size reduction on computed tomography (CT) scans. 1 Our present studies, therefore, suggest that MAb A7 may be useful as a carrier for targeting chemotherapy.
Acknowledgment.
This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for the Comprehensive 10-Year Strategy for Cancer Control.
A
B
C
D
Fig. 2. SDS-PAGE and immunoblotting of the MAb A7defined antigen. NP-40 cell lysates and spent media were subjected to immunoblotting followed by SDS-PAGE. The band was detected after immunoperoxidase staining by MAb A7 (10pg/ml). A n A7-defined antigen with a molecular weight of approximately 42,000 was detected from HPC-YS and WiDr cell lysates and the spent medium of HPC-Y9, but not from the KB-2 cell lysate. (Lane A, HPC-YS cell lysate; B, WiDr cell lysate; C, spent medium of HPC-Y9; D, KB-2 cell lysate)
small amounts, in the sera of pancreatic carcinoma patients because the antigen was recognized by MAb A7 in the medium of only one cell line, HPC-Y9. However, MAb A7 reacted with the pancreatic carcinoma tissues at a high rate, suggesting that MAb A7 can reach the pancreatic cancer tissues without binding to the antigen in serum. Furthermore, in the buffered formalin-fixed and paraffin-embedded sections of HPCYS, the a n t i g e n r e c o g n i z e d by M A b A 7 s e e m e d to be c o n f i n e d to the cell surface. M o r e o v e r , the a n t i g e n was m a i n l y d e t e c t e d o n the cell surface in the acetone-fixed s m e a r s p e c i m e n s of the positive-staining p a n c r e a t i c
References 1. Takahashi T, Yamaguchi T, Kitamura K, Suzuyama H, Honda M, Hashimoto Y (1988) Clinical application of monoclonal antibody-drug conjugates for immunotargeting chemotherapy of colorectal carcinoma. Cancer 61:881-888 2. Gold P, Freedman SO (1965) Demonstration of tumor specific antigens in human colon carcinoma by immunological tolerance and absorption technique. J Exp Med 121:439-462 3. Magnani JL, Steplewski Z, Koprowski H, Ginsburg V (1983) Identification of the gastrointestinal and pancreatic cancerassociated antigen detected by MAb 19-9 in the sera of patients as a mucin. Cancer Res 43:5489-5492 4. Gelder FB, Reese CJ, Moossa AR, Hall T, Hunter R (1978) Purification, partial characterization, and clinical evaluation of a pancreatic oncofetal antigen. Cancer Res 38:313-324 5. Browitz MJ, Tuck FL, Sindelar WF, Fernsten PD, Metzgar RS (1984) Monoclonal antibodies against human pancreatic adenocarcinoma: Distribution of DU-PAN-2 antigen on glandular epithelia and adenocarcinoma. J Natl Cancer Inst 72:999-1003 6. Fukushi Y, Kannagi R, Hakomori S (1985) Location and distribution of difocoganglioside in normal and tumor tissues defined by its monoclonal antibody FH-6. Cancer Res 45:37113717 7. Metzgar RS, Gaillard MT, Levine SJ, Tuck FL, Bossen EH, Borowitz MJ (1982) Antigen of human pancreatic adenocarcinoma cells defined by murine monoclonal antibodies. Cancer Res 42:601-606
356 8. Klavince JV (1981) Tumor markers of pancreatic carcinoma. Cancer 47:1957-1601 9. Fukuda K (1985) The study of targeting chemotherapy against the gastrointestinal cancer (the preparation of anticancer drug - monodonal antibody conjugate and the investigation about its biological activity). Akita J Med 12:451-468 10. Yamaguchi N, Kawai K (1986) Acid protease from human pancreatic carcinoma cell line HPC-YT into serum-free, chemically defined medium. Cancer Res 46:5353-5359 11. Siroeda O, Yamaguchi N, Kawai K (1987) Stimulation of low density lipoprotein receptor activity by conditioned medium from human cancer cell line. Cancer Res 47:4630-4633 12. Kotanagi H, Takahashi T, Masuko Y, Hashimoto Y, Koyama K (1986) A monoclonal antibody against human colon cancers. Tohoku J Exp Med 148:353-360 13. Su-Ming Hsu, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase technique. J Histochem Cytochem 29:577-580 14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 15. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci 76:4350-4353 16. Marianne J, Clausen PP, Susanne S (1980) The effect of fixation
E. Otsuji et al.: Expression of the A7 Antigen and tripsinization on the immunohistochemical demonstration of intracellular immunogroblin in paraffin embedded material. Acta Path Microbiol Scand Sect 88:369-376 17. Allum WIF, Stokes HJ, Macdonald F, Fielding JWL (1986) Demonstration of carcinoembryonic antigen (CEA) expression in normal, chronically inflamed, and malignant pancreatic tissue by immunochemistry. J Clin Pathon 39:610-614 18. Inufusa H (1988) Development of spontaneous lung metastasis (in Japanese). Nippon Geka Gakkai Zasshi (J Jpn Surg Soc) 89:1075-1082 19. Chin JK, Rong GH, Scharff JE, Sindelar WF (1986) Gastrointestinal carcinoma-associated antigen defined by a murine monodonal antibody. J Natl Cancer Inst 77:599-603 20. Zenon S, Toong H, Herlyn M, Koprowski M (1981) Release of monoclonal antibody-defined antigens by human colorectal carcinoma and melanoma cells. Cancer Res 41:2723-27 21. Ban T, Imai K, Takai Y, Ibayashi Y, Hirai K, Sugiyama T, Endo T, Yachi A (1988) Comparison between two new tumor markers "Sialyl SSEA-1 antigen" and "YH 206 antigen" in sera of pancreatic cancer patient. Suizou 3:507-512 22. Kitamura K, Takahashi T, Yamaguchi T, Yokota T, Noguchi A, Amagai T, Imanishi J (1989) Immunochemical characterization of the antigen recognized by the murine monodonal antibody A7 against human colorectal cancer. Tohoku J Exp Med 157:83-9~
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SURC~RYTODAY © Springer-Verlag 1992
Expression of the Cell Surface Antigen Detected by the Monoclonal Antibody A7 in Pancreatic Carcinoma Cell Lines EIGO OTSUJI, 1 TOSHIHARU YAMAGUCHI,1 NOZOMI YAMAGUCHI,2 KUNIHIKO KOYAMA,3 JIRO IMANISHI,2 NOBUKI YAMAOKA,2 and TOSHIO TAKAHASHIt 1The First Department of Surgery, and the Departments of 2Microbiology and 3Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji Agaru Kajiicho 465, Kamigyo-ku, Kyoto 602, Japan
Abstract: In a previous study, we used a murine monoclonal antibody, AT, against human colon carcinoma as a drugcarrier to treat colorectai cancer. ~ In the present study, we found that MAb A7 also reacted immunohistochemically with 73% of human pancreatic carcinoma cell lines, with the A7 antigen mainly being detected on the cell surface. However, the A7 antigen was found in only 9% of the spent media of these human pancreatic carcinoma call lines by ELISA. On the other hand, the positive incidence of CA19-9, POA, ferritin, CEA, DU-PAN-2 and SLX in those spent media was 100%, 64%, 64%, 55%, 55% and 36%, respectively. These results suggest that the A7 antigen may only rarely be shed into the sera of pancreatic cancer patients, in which case MAb A7 could be a suitable drug-carrier in targeting chemotherapy for pancreatic cancer patients. Key Words: monoclonal antibody A7, pancreatic tumor
marker, pancreatic carcinoma cell line, immunotargeting chemotherapy
Introduction
A number of antigens associated with pancreatic carcinoma, such as carcinoembrionic antigen (CEA), pancreatic oncofetal antigen (POA), ferritin, DUPAN-2, CA19-9 and sialyl Le x antigen (SLX), have been identified using polyclonal or monoclonal antibodies. 2-8 Although most antibodies induced by these antigens were not primarily produced by immunization with pancreatic carcinomas, they have been detected in pancreatic carcinoma by analyses using various kinds of
Reprint requests to: E. Otsuji (Received for publication on Jan. 8, 1991; accepted on May 1, 1992)
monoclonal antibodies. Antibodies to these antigens have been used clinically for the serological diagnosis of pancreatic cancer since large amounts of these antigens are shed or released from cancer cells into serum. For targeting chemotherapy, however, a large number of antigens in serum is disadvantageous because the administered antibody, conjugated to an anticancer agent, binds to the antigen in the serum before it arrives at the tumor. In our laboratory, the monoclonal antibody A7 (MAb) was produced against a human colon carcinoma and conjugated covalently with the anticancer antibiotic, neocarzinostatin (NCS; Kayaku, Japan). The monoclonal antibody-drug conjugate, A7-NCS, 9 was then used clinically to treat patients with colorectal carcinoma. 1 There is little information available on the treatment of pancreatic cancer with a monoclonal antibody. A7NCS might be useful for the targeting chemotherapy of pancreatic cancer if MAb A7 reacts with pancreatic carcinoma at a high rate and if large amounts of the antigen are not shed from pancreatic carcinoma cells. We describe herein the reactivity of MAb A7 with 11 human pancreatic carcinoma cell lines and the cytochemical and immunological characterization of the monoclonal antibody A7.
Materials and Methods
Cell Lines
The following cell lines were used: 11 human pancreatic carcinoma cell lines, being HPC-YO, HPC-Y1, HPCY5, HPC-Y9, HPC-Yll, HPC-Y15, HPC-Y19, HPCY25, HPC-YP, HPC-YS and HPC-YT, a human colonic carcinoma cell line, being WiDr, and a human laryngeal carcinoma cell line, being KB-2. All the
352 pancreatic carcinoma cell lines were established from ductal cell adenocarcinoma except for HPC-YO, which was established from acinal cell carcinoma. All the human pancreatic carcinoma cell lines were established by Dr. Yamaguchi of the Kyoto Prefectural University of Medicine, Japan, who also maintained and kindly donated the other cell lines. These cell lines were cultured in protein-free Ham's F-12 medium (Flow Laboratories, USA) without any other supplements, as described previously. 1°'~1
Spent Media of the Human Pancreatic Carcinoma Cells The serum-free medium was changed every 4 days and the spent medium collected aseptically. After the cellular debris was removed by centrifugation, the spent medium was stored at 4°C and concentrated by ultrafiltration using a PTGC membrane with a molecular weight cut off of 10,000 (Nippon Millipore, Japan) at 0°C. 1°'11 Finally, the concentrated spent medium was dialyzed against distilled water and lyophilized.
Monoclonal Antibody A 7 MAb A7 was produced by a hybridoma obtained after fusing spleen cells from a mouse immunized with human colon adenocarcinoma, Colon-6, with murine myeloma P3.X63.Ag8.653 cells, as described previously, lz MAb A7 was purified by the Protein A method.
Immunoperoxidase Staining of the Human Pancreatic Carcinoma Cell Lines Cultured human pancreatic carcinoma cells detached with phosphate-buffered saline (PBS) containing 2 mM EDTA were washed in PBS three times and smear specimens prepared. After fixation with cold acetone for five min, immunoperoxidase staining was performed by the avidin-biotin-peroxidase complex (ABC) method (Vector Labs, USA). 13 The carcinoma cells were then stained with MAb A7 in PBS (10pg/ ml) with the monoclonal antibodies of DU-PAN-2 (Kyowa medex, Japan), diluted to 1/300 with PBS, CA19-9 (CIS Kit) and SLX (Otsuka Assay Lab., Japan) in a spent medium of FH-6, and with the polyclonal antibodies of CEA (DAKO, Denmark, 1/500), POA (provided by Dr. Takami of the University of Teikyo, 1/500) and ferritin (Miles, Denmark, 1/500). Finally, the specimens were developed with diaminobenzidine for color, then counterstained with hematoxylin.
E. Otsuji et al.: Expression of the A7 Antigen
Cellular Localization of the Antigen Recognized by MAb A7 After the HPC-YS cells were harvested with EDTA and washed in PBS three times, they were fixed with 10% buffered formalin in PBS for one hour at room temperature. Following dehydration, they were embedded in paraffin and 6-~tm serial sections were cut. After these sections were deparaffinized and processed in alcohol by standard techniques, immunoperoxidase staining was performed by the ABC method, after which the nucleus was counterstained with hematoxylin. Normal mouse IgG was used for the control staining.
ELISA Against a Spent Media of Human Pancreatic Carcinoma Cell Lines The ELISA was performed against the spent media of 11 human pancreatic carcinoma cell lines. Thirtyfive ~tg of freeze-dried serum-free spent medium was suspended in carbonate-bicarbonate buffer (pH 9.6) and seeded in 96-well microtiter plates (Flow Laboratories, USA). After overnight incubation at room temperature, the wells were treated with 25% Block Ace (Dainippon Kagaku) in PBS. Following removal of the Block Ace, 50 pl of MAb A7 in PBS (10 ~tg/ml) was added and the wells were incubated for 1 hour at room temperature. After three washes with 0.05 per cent Tween 20-PBS, the wells were converted with peroxidase-labeled goat anti-mouse IgG + IgM (Kirkegraard and Perry Laboratories, USA). After five rinses with 0.05% Tween 20-PBS, 50pl of a substrate solution containing diethylbenzothiazolin (ABTS, Kirkegraard and Perry Laboratories, USA) was added to each well. The absorbance was read at 414nm by an automatic immunoreader (Bio Rad, USA). As a control, MAb A7 was replaced by 10 ~tg of normal mouse IgG (Funakoshi, Japan). Using the same method, ELISA was performed with the monoclonal antibodies of DU-PAN-2, CA19-9 and SLX, and the polyclonal antibodies of CEA, POA and ferritin at the same concentrations used in immunoperoxidase staining, with appropriate antisera as a control.
Biochemical Characterization of the Antigen Detected by MAb A7 on the Cultured Cells and in Their Spent Media HPC-YS cultured cells (5 × 106) were solubilized with 500 ~tl of the extraction buffer (10 mM Tris-HC1 pH.7.4, 150mM NaC1, 0.5% Nonidet-P40). After the mixture was incubated for 30 min at 0°C and centrifuged at 10,000 × g for 20 rain, the supernatant was added to the same amount of the sample buffer con-
E. Otsuji et al.: Expression of the A7 Antigen
353
Table 1. Reactivity of the antibodies to human pancreatic carcinoma cell lines
Anti-CEA Anti-POA Anti-ferritin Anti-DU-PAN-2 Anti-CA19-9 Anti-SLX A7
HPC -YO
HPC -Y1
HPC -Y5
HPC -Y9
HPC -Yll
HPC -Y15
HPC -Y19
HPC -Y25
HPC -YP
HPC -YS
HPC -YT
Total (%)
+ + + + + -
+ + + + -
+ + + + + + +
+ + + + + + +
+ + + + + +
+ + + + + +
+ + + + + + -
+ + + + +
+ + + + +
+ + + + + + +
+ + + +
11/11 (100) 9/11 (82) 10/11 (91) 6/11 (55) 11/11 (100) 6/11 (55) 8/11 (73)
taining 2% sodium dodecyl sulfate (SDS) without mercaptoethanol. The spent medium of the cultured H P C - Y 9 cells (200 gg) was mixed with 10 gl of sample buffer containing SDS without mercaptoethanol. After being boiled for 3 min, the samples were analyzed by electrophoresis in 4 - 2 0 % gradient SDS-polyacrylamide gels (Daiichi Pure Chemicals, Japan) according to the method of Laemmli. 14 Protein bands were then electrophoretically transferred to a nitrocellulose m e m b r a n e (BA85 Schleicher and Schuell, West G e r m a n y ) as described previously by Towbin 15 (LKB Electrophoretic Transfer Kit). Electroblotting was p e r f o r m e d at 60 m A for 2 hours. After the transfer, the nitrocellulose m e m brane was rinsed in Tween 20-PBS and coated with Block Ace overnight at 4°C. The m e m b r a n e was then incubated with 10 gg/ml of M A b A7 in PBS for 1 h at r o o m t e m p e r a t u r e and stained by the A B C method. As a control, the m e m b r a n e was incubated with normal mouse IgG. The lysates of the h u m a n colon carcinoma cell line WiDr, or the h u m a n larynx squamous carcinoma cell line KB-2 were electrophoresed at the same time as positive or negative controls.
Results
Immunocytochemical Reactivity of MAb A7 with the Human Pancreatic Carcinoma Cell Lines Table 1 shows the reactivity of M A b A7 with the h u m a n pancreatic carcinoma cell lines by the immunoperoxidase method in comparison with that of antibodies against other well-known t u m o r markers. M A b A7 detected the antigen in 8 of the 11 h u m a n pancreatic carcinoma cell lines (73%), namely, H P C , Y 5 , HPC-Y9, H P C - Y l l , HPC-Y15, HPC-Y25, H P C - Y P , H P C - Y S and H P C - Y T . Almost 100% of the cells in these cell lines were positively stained, except for the H P C - Y P cells, of which about 50% were stained with M A b A7. Three of the 11 cell lines (27%), being H P C - Y O , HPC-Y1 and HPC-Y19, were not stained at all by M A b A7. The anti-CEA and anti-
CA19-9 antibodies both reacted with all 11 cell lines, the anti-ferritin antibody with 10 (91%), and the antiP O A antibody with 9 (82%). Six cell lines (55%) were stained by both the anti-DU-PAN-2 and anti-SLX antibodies. All the antibodies reacted with HPC-Y5, H P C - Y 9 and HPC-YS, while HPC-Y19 stained with all the antibodies except for M A b A7.
Cellular Localization of the Antigen Detected by MAb A7 As shown in Fig. 1, the antigen was mainly detected by M A b A7 on the cell surface of HPC-YS cells with immunoperoxidase staining. The cytoplasm was only slightly stained, and the mouse I g G control did not detect the antigen.
Reactivity Against the Spent Media of the Human Pancreatic Carcinoma Cell Lines by ELISA The presence of these tumor-associated antigens in tissue culture media as determined by E L I S A is shown in Table 2. M A b A7 detected the antigen in the medium of only 1 of the 11 cell lines (9%). On the other hand, CA19-9 antigen wa~ detected in the spent media of all the cell lines (100%). P O A and ferritin were each detected in 7 (64%), and C E A and D U - P A N - 2 each in 6 spent media (55%). The positive incidence of SLX was lowest among all the well-known t u m o r markers (36%).
Analysis of the A 7 Antigen by SDS-Polyacrylamide Gel Electrophoresis and Western Blotting To determine the molecular weight (MW) of the antigen recognized by M A b A7, SDS-polyacrylamide gel electrophoresis and immunoblotting were e~rformed. In the lane where the lysates of HPC-YS and W i D r were applied, M A b A7 bound to a c o m p o n e n t with M W 42,000. In the lane with H P C - Y 9 spent medium, the binding of M A b A7 was directed against the band at M W 42,000. In the lane with the squamous
354
E. Otsuji et al.: Expression of the A7 Antigen
Fig. 1. Cellular localization of the M A b A7-defined antigen was observed by immunoperoxidase staining of formalin-fixed paraffinembedded sections of the human pancreatic carcinoma cell line, HPCYS. The nucleus was counterstained with hematoxylin and the antigen mainly detected on the cell membrane by M A b A7 (10~tg/ml) (left). Positive staining was not seen with normal mouse IgG (101ag/ml)
(right)
Table 2. Reactivity of the antibodies to the spent media of human pancreatic carcinoma cell lines
Anti-CEA Anti-POA Anti-ferritin Anti-DU-PAN-2 Anti-CA19-9 Anti-SLX A7
HPC -Y0
HPC -Y1
HPC -Y5
HPC -Y9
HPC -Yll
HPC -Y15
HPC -Y19
HPC -Y25
HPC -YP
HPC -YS
HPC -YT
+ -
+ + + -
+ + + + -
+ + + + + + +
+ + + + + .
+ + + + -
+ + + + + .
+ + + + -
+ -
+ + + + + +
+ + -
cell c a r c i n o m a cell line, K B - 2 , M A b A 7 d i d n o t b i n d to a n y c o m p o n e n t (Fig. 2). N o r m a l m o u s e I g G d i d n o t stain. W h e n 2 - m e r c a p t o e t h a n o l was a d d e d to t h e s a m p l e b u f f e r no b a n d was s e e n in a n y l a n e ( d a t a n o t shown).
Discussion This p a p e r d e s c r i b e s t h e r e a c t i v i t y o f t h e m o n o c l o n a l a n t i b o d y A 7 with 11 h u m a n p a n c r e a t i c c a r c i n o m a cell lines a n d t h e i r s p e n t m e d i a . W e u s e d a c e t o n e - f i x e d s p e c i m e n s b e c a u s e f o r m a l i n fixation a n d paraffin e m b e d d i n g s o m e t i m e s e x e r t a s t r o n g influence o n t h e p r e s e r v a t i o n o f antigenicity.16'17 M A b A 7 r e a c t e d with 7 3 % o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines. G e n e r a l l y , t h e r e a c t i v i t y o f an a n t i b o d y to a c a r c i n o m a cell line is l o w e r t h a n t h a t to c a r c i n o m a tissue which consists o f h e t e r o g e n e o u s t u m o r cells. 18 H o w e v e r , t h e p o s i t i v e i n c i d e n c e of t h e 6 w e l l - k n o w n t u m o r m a r k e r
.
.
.
.
.
Total (%) 6/11 7/11 7/11 6/11 11/11 4/11 1/11
(55) (64) (64) (55) (100) (36) (9)
a n t i g e n s in o u r h u m a n p a n c r e a t i c c a r c i n o m a cell lines was s i m i l a r to t h a t f o u n d b y o t h e r i n v e s t i g a t o r s using p a n c r e a t i c c a r c i n o m a tissues. 4'5's T h e p o s i t i v e inc i d e n c e o f M A b A 7 was l o w e r t h a n t h a t o f t h e antib o d i e s a g a i n s t CEA, C A 1 9 - 9 , ferritin a n d P O A , b u t h i g h e r t h a n t h a t o f t h e a n t i b o d i e s against D U - P A N - 2 and SLX. T h e stain!ng h e t e r o g e n e i t y o f H P C - Y P b y M A b A 7 was s i m i l a r to t h a t d e s c r i b e d in o t h e r studies. 5'19 This h e t e r o g e n e i t y m a y b e r e l a t e d to t h e stage of different i a t i o n o f t h e cells o r t h e i r p h a s e in t h e cell cycle. E L I S A was p e r f o r m e d to d e t e c t a n t i g e n s in t h e m e d i a o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines. W e u s e d a s e r u m - f r e e m e d i u m b e c a u s e it was m o r e s u i t a b l e for t h e a n t i g e n assay. 2° T h e p o s i t i v e i n c i d e n c e o f t h e w e l l - k n o w n t u m o r m a r k e r s in t h e s p e n t m e d i a o f t h e h u m a n p a n c r e a t i c c a r c i n o m a cell lines was also s i m i l a r to t h a t in t h e s e r a o f p a n c r e a t i c c a r c i n o m a p a t i e n t s in p r e v i o u s r e p o r t s . 4'21 T h e a n t i g e n r e c o g n i z e d b y M A b A 7 a p p e a r s to b e r a r e , o r p r e s e n t in o n l y v e r y
E. Otsuji et al.: Expression of the A7 Antigen
355
carcinoma cell lines (data not shown). Although this method is suitable for detecting the cytoplasmic antigen, the cytoplasm was stained very weakly by MAb A7. Generally, it is necessary for the antigen to be localized on the cell surface in order to detect the tumor cell in vivo. These results suggest that MAb A7 may be useful for targeting chemotherapy. Using SDS-PAGE and immunoblotting, we showed that the epitope defined by MAb A7 in the lysate of the HPC-YS cell membrane and in the spent medium of HPC-Y9 cells was located on a 42K dalton molecule. Identical results were obtained using the human colon carcinoma cell line, WiDr. This is in accordance with a previous report characterizing the antigen defined by MAb A7 on the human colon carcinoma cell line, SW 1116. 22 In the presence of mercaptoethanol in the sample buffer, however, the antigen was not detected. MAb A7 has been conjugated to NCS, and MAb A7NCS conjugate has been used to treat patients with colorectal cancer. Patients receiving the conjugate did not experience serious adverse effects, and some showed such therapeutic benefits as pain relief and tumor size reduction on computed tomography (CT) scans. 1 Our present studies, therefore, suggest that MAb A7 may be useful as a carrier for targeting chemotherapy.
Acknowledgment.
This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for the Comprehensive 10-Year Strategy for Cancer Control.
A
B
C
D
Fig. 2. SDS-PAGE and immunoblotting of the MAb A7defined antigen. NP-40 cell lysates and spent media were subjected to immunoblotting followed by SDS-PAGE. The band was detected after immunoperoxidase staining by MAb A7 (10pg/ml). A n A7-defined antigen with a molecular weight of approximately 42,000 was detected from HPC-YS and WiDr cell lysates and the spent medium of HPC-Y9, but not from the KB-2 cell lysate. (Lane A, HPC-YS cell lysate; B, WiDr cell lysate; C, spent medium of HPC-Y9; D, KB-2 cell lysate)
small amounts, in the sera of pancreatic carcinoma patients because the antigen was recognized by MAb A7 in the medium of only one cell line, HPC-Y9. However, MAb A7 reacted with the pancreatic carcinoma tissues at a high rate, suggesting that MAb A7 can reach the pancreatic cancer tissues without binding to the antigen in serum. Furthermore, in the buffered formalin-fixed and paraffin-embedded sections of HPCYS, the a n t i g e n r e c o g n i z e d by M A b A 7 s e e m e d to be c o n f i n e d to the cell surface. M o r e o v e r , the a n t i g e n was m a i n l y d e t e c t e d o n the cell surface in the acetone-fixed s m e a r s p e c i m e n s of the positive-staining p a n c r e a t i c
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